Prevention and treatment of inflammatory conditions

ABSTRACT

The present embodiments relate to methods for the prevention and treatment of inflammatory conditions such as alcoholic liver disease (ALD). More specifically the present embodiments relate to the prevention and treatment of ALD through the administration of an Retinoic Acid Receptor (RAR) agonist. Some embodiments relate to use of tazarotene in the prevention and treatment of alcohol-induced liver injury, alcohol-related liver disease, fatty liver disease, hepatic steatosis, alcoholic hepatitis or alcoholic cirrhosis.

This application is continuation of U.S. patent application Ser. No.15/913,541, filed Mar. 6, 2018, which is a continuation of U.S. patentapplication Ser. No. 14/128,566, filed Feb. 14, 2014, now U.S. Pat. No.9,949,996, which is a national stage entry, filed under 35 U.S.C. § 371,of International Application No. PCT/US2012/043875, filed Jun. 22, 2012,which claims priority to U.S. Provisional Application No. 61/501,139,filed Jun. 24, 2011, the entire contents of each are incorporated byreference herein in their entireties.

BACKGROUND Field

The present embodiments relate to compositions and methods formodulating Retinoic Acid Receptor (RAR) in the prevention and treatmentof inflammatory conditions of the liver.

Description of the Related Art

Excessive alcohol use is a major cause of liver disease in the Westernworld. Evidence of liver injury is observed in individuals who consumefour or more alcoholic drinks a day (four 12 ounces beers, four glassesof wine, or four ounces of hard liquor for men or half that quantity forwomen). Although how alcohol damages the liver is not fully understood,chronic alcohol consumption results in the secretion of pro-inflammatorycytokines (TNF-alpha, IL6 and IL8), oxidative stress, lipidperoxidation, and acetaldehyde toxicity, resulting in inflammation,apoptosis and eventually fibrosis of liver cells.

Alcoholic liver disease (ALD) has three main phases: alcoholic fattyliver disease, alcoholic hepatitis, and cirrhosis. Alcoholic fatty liverdisease, which is characterized by an accumulation of fatty acids in theliver, is usually asymptomatic and reversible if the individual abstainsfrom alcohol for a couple of weeks. In severe cases, weakness, nausea,abdominal pain, loss of appetite, and malaise may be experienced.Although most heavy drinkers exhibit some level of fatty liver disease,in some cases, heavy drinking need only have occurred daily over aperiod of than less than a week, only one in five heavy drinkersdevelops alcoholic hepatitis, and one in four develops cirrhosis.Alcoholic hepatitis is characterized by the inflammation of hepatocytesand is generally reversible by abstinence. Cirrhosis, which ischaracterized by inflammation, fibrosis (cellular hardening) and damagedmembranes preventing detoxification of chemicals in the body, ending inscarring and necrosis, is generally irreversible.

The cellular and molecular mechanisms underlying different phases ofliver tissue damage in alcoholic liver disease are poorly understood.Although progress has been made in several areas, an effectivetherapeutic approach to halt this disease is still lacking. This is inpart owing to the fact that the liver is a unique organ immunologicallyas well as anatomically. For example, while hepatic parenchymal cellshave metabolic functions, non-parenchymal cells perform immunologicalfunctions. In addition to parenchymal hepatocytes, the liver containsseveral non-parenchymal cells such as LSEC, Kupffer cells, dendriticcells, NK cells and NKT cells that all can participate in immunity. Howthe immune response is orchestrated to give either tolerance or immunityto alcohol-induced damage is not known.

SUMMARY

The present embodiments generally relate to methods and compositions formodulating the innate immune system to prevent and treat tissue damageassociated with inflammatory conditions. For example, severalembodiments described herein relate to the prevention and treatment ofalcoholic liver disease (ALD) by modulating the innate immune system.

Several embodiments described herein relate to methods and compositionsfor manipulating the activities of type I NKT cells and type II NKTcells, interactions between type I and type II NKT cells, and theirinteractions with other liver cells in order to treat, alleviate orprevent inflammation-associated injury to the liver. In someembodiments, the inflammation-associated injury is an alcohol-inducedliver injury. In some embodiments, the alcohol-induced liver injury isalcohol-related liver disease, fatty liver disease, hepatic steatosis,alcoholic hepatitis or alcoholic cirrhosis.

Several embodiments relate to a method of preventing, mitigating ortreating inflammatory induced liver damage following excessive alcoholconsumption by inhibiting type I NKT cell activity. In some embodiments,pro-inflammatory type I NKT cell activity is inhibited by one or moreRAR agonists selected from the group consisting of ATRA, retinol,9-cis-RA or 13-cis-RA, tretinoin, AM580, AC55649, CD1530 and Tazarotene.In some embodiments, pro-inflammatory type 1 NKT cell activity isinhibited by one or more polyolefinic retinoids, such as isoretinoin andacitretin. In some embodiments, pro-inflammatory type I NKT cellactivity is inhibited by one or more RAR agonists selected from thegroup consisting of etretinate, acitretin and isotretinoin. Severalembodiments relate to the inhibition of pro-inflammatory type I NKT cellactivity by tazarotene, tazarotenic acid or a mixture thereof. In someembodiments, pro-inflammatory type I NKT cell activity is inhibited byactivating type 2 NKT cells. In some embodiments, inflammatory type INKT cell activity is inhibited by one or more sulfatides.

Some embodiments of the present disclosure are related to the innateimmune mechanisms leading to liver injury following, related to orcaused by alcohol consumption. Some embodiments relate to manipulatingthe interactions among these cells which naturally provide tolerance togut-derived or metabolite-derived antigens and at the same time provideimmunity against non-self identified pathogens.

Some embodiments relate to a combination therapy approach targeting bothtype I and type 11 NKT cells for the development of an effectivetherapeutic to treat, prevent or mitigate tissue damage associated withinflammatory conditions. In some embodiments, an RAR agonist is used todirectly inhibit type 1 NKT cell activity and a sulfatide is used toactivate type II NKT cell activity.

Some embodiments relate to the prevention and treatment of tissue damageassociated with ALD through the administration of all trans retinoicacid (ATRA). Several embodiments relate to use of ATRA in the preventionand treatment of alcohol-induced liver injury, alcohol-related liverdisease, fatty liver disease, hepatic steatosis, alcoholic hepatitis oralcoholic cirrhosis.

Some embodiments relate to the prevention and treatment of tissue damageassociated with ALD through the administration of tazarotene,tazarotenic acid or a mixture thereof. Several embodiments relate to useof tazarotene, tazarotenic acid or a mixture thereof in the preventionand treatment of alcohol-induced liver injury, alcohol-related liverdisease, fatty liver disease, hepatic steatosis, alcoholic hepatitis oralcoholic cirrhosis.

Some embodiments relate to the prevention and treatment of tissue damageassociated with inflammatory through the administration of one of moresulfatides. Several embodiments relate to use of sulfatide in theprevention and treatment of alcohol-induced liver injury,alcohol-related liver disease, fatty liver disease, hepatic steatosis,alcoholic hepatitis or alcoholic cirrhosis. In some embodiments, thesulfatide has following chemical structure:

wherein R₁ is selected from the group consisting of a bond, a hydrogen,a C to C₃₀ alkyl, C₁ to C₃₀ substituted alkyl, a C₁ to C₃₀ alkenyl, a C₁to C₃₀ substituted alkenyl and a C₅ to C₁₂ sugar; R₂ is selected fromthe group consisting of a hydrogen, a hydroxy group, a methoxy group,and an alkoxy group; R₃ is selected from the group consisting of ahydrogen, a hydroxy group, a methoxy group, an ethoxy group, and analkoxy group; R₄ is selected from the group consisting of a hydrogen, ahydroxy group and an alkoxy group; R₅ is selected from the groupconsisting of a hydrogen, a hydroxyl, a carbonyl, an alkoxy and a bond;R₆ is selected from the group consisting of a C₁ to C₄₀ alkyl, a C₁ toC₄₀ substituted alkyl, a C₁ to C₄₀ alkenyl, a C₁ to C₄ substitutedalkenyl and a C₁ to C₄₀ alkynl; R₇ is selected from the group consistingof a C₁ to C₄ alkyl, a C₁ to C₄₀ substituted alkyl, a C₁ to C₄₀ alkenyl,a C₁ to C₄₀ substituted alkenyl and a C₁ to C₄₀ alkynl; and R₈ isselected from the group consisting of a hydrogen, a hydroxyl group, acarbonyl, an alkoxy group and a bond. In some embodiments, the sulfatidehas following chemical structure:

Some embodiments relate to the regulation of type I NKT cells byactivated sulfatide-reactive type II NKT cells. Several embodimentsrelate to the regulatory role of activated sulfatide-reactive type IINKT cells on type I NKT cells in mediating protection from autoimmunedisease and suppression of anti-tumor immunity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a depicts a methodology by which sulfatide's effect on type I NKTin the liver is determined. FIG. 1 b shows representative data of twoindependent experiments measuring cell proliferation in response to anin vitro challenge with αGalCer in the presence or absence of 10 ng/mlIL-2. FIG. 1 c shows the results of intracytoplasmic staining ofsulfatide/CD1d-tetramer+ and tetramer− populations in liver to identifyIFN-γ+ cells following injection with 20 μg sulfatide. Numbers abovebracket indicate % positive cells in PBS or sulfatide-injected mice.FIG. 1 d shows micrographs (×200) of representative hematoxylin andeosin (H&E)-stained liver sections taken from mice at 12-96 hoursfollowing treatment with ConA alone (top row) or ConA plus bovine brainsulfatide (bottom row). The bottom left shows a representativemicrograph of an H&E-stained liver section from a control mouse injectedwith sulfatide alone. FIG. 1 e shows graphs depicting alanine aminotransferase (ALT) and aspartate amino transferase (AST) levels inIL-12p40+/+ mice at different time points after ConA (filled symbols) orConA plus sulfatide (open symbols) injection. Values are mean SD of 5mice per group. P<0.001.

FIG. 2 a shows flow plots of Gr-1/CD11b expressing cells isolated fromcephalad liver lobes of wild type (WT), Jα18−/− mutant andsulfatide-treated WT mice subjected to shame surgery or ischemicreperfusion injury (IRI). The boxes show the Gr-1^(high) and Gr-1^(int)populations analyzed in FIG. 2 b . Numbers next to boxes indicatepercent positive cells among liver leukocytes. FIG. 2 b shows bar graphssummarizing the flow cytometric analysis. The left panel depicts the %and the right panel depicts the absolute numbers of the Gr-1^(high) andGr-1^(int) populations. Values are mean t SEM. * p<0.005. FIG. 2 c showsa graph depicting the % IFN-γ+αGalCer/CD1d-tetramer+ cells in the liversof IRI, sham and sulfatide-treated IRI mice. Values are mean±SEM.P<0.01.

FIG. 3 depicts a model for sulfatide mediation of Type I and Type II NKTcell activity. According to the model, sulfatide promotes activation oftype II NKT cells, which in turn inactivate type I NKT cells. Inhibitionof type I NKT cells is exemplified by inhibition of IFN-γ secretionresulting in reduced hepatic recruitment of myeloid cells, granulocytesand consequently in reduced hepatocyte and endothelial cell injury.

FIG. 4 show representative micrographs of H&E stained tissue fromcephalad liver lobes of mice subjected to 90 min of hepatic ischemiafollowed by 24 hrs of reperfusion (IRI) (top row) and mice subjected tosham surgery (sham) (bottom row) at 100× magnification. From left toright, the panels show tissue from untreated WT, untreated Jα18^(−/−),sulfatide-treated WT, and sulfatide-treated Jα18^(−/−) mice.

FIG. 5 shows graphs summarizing flow cytometry analysis of liver MNCsisolated from groups (4 in each) of male BU6 WT or Jα18−/− micefollowing 5 weeks of feeding with a liquid Lieber-Decarli dietcontaining 5% ethanol or control diet containing similar number ofcalories. P values <0.005. Accumulation of activated type I NKT cellsand CD11b+Gr-1+ myeloid cells was observed in the livers of WT but notJα18−/− mice.

FIG. 6 shows a bar graph depicting serum ALT levels in WT (B6) andJα18-mice injected (i.p.) with 20 micrograms/mouse sulfatide (d-1 & d10)(bars shaded with horizontal lines (Sulfatide)), 0.3 milligrams/animalATRA (d6 through d10) (bars shaded with vertical lines (atRA)) orvehicle/DMSO (solid shaded bars (EtOH)) and fed Lieber-DeCarli liquiddiet containing alcohol. The control groups (unshaded bars) were fed anisocaloric control diet. P values <0.05.

FIG. 7 show representative micrographs (×200) of liver tissue harvestedfrom vehicle/DMSO, sulfatide and ATRA treated mice following chronicplus binge ethanol feeding. Liver tissue from vehicle/DMSO mice showsevidence of hepatic steatohepatitis (fatty liver disease), while livertissue obtained from sulfatide and ATRA treated mice appears relativelynormal.

FIG. 8 a shows a bar graph depicting proliferation (as measured by[³H]-thymidine incorporation) of freshly isolated type I NKT spleniccells incubated in αGalCer alone, ATRA alone or in αGalCer withincreasing levels of ATRA. FIG. 8 b shows a graph depicting the level ofIL-2 cytokine secretion by a type I NKT cell (Hy1.2) in response toαGalCer alone or αGalCer and increasing ATRA levels. FIG. 8 c showsgraphs depicting the levels of IFN-γ, IL-4, IL-13, IL-6, IL-10 and IL-12cytokine secretion by liver type I NKT cells harvested from miceinjected with DMSO or ATRA and incubated with αGalCer in vitro. The datashown is representative of 2 individual experiments.

FIG. 9 a shows representative micrographs (×200) of H&E stained livertissue harvested from male BU6 and Jα18−/− mice following chronic (10days 5% ethanol in liquid Lieber-DeCarli diet) or chronic plus bingefeeding (ten days of chronic feeding of liquid diet followed by a singlegavage of 5 g/kg ethanol). FIG. 9 b shows graphs depicting serum ALTlevels in control or ethanol fed male BU6 or Jα18−/− mice followingchronic or chronic plus binge feeding.

FIG. 10 shows a bar graph depicting proliferation (as measured by[³H]-thymidine incorporation) of freshly isolated myelin basic proteinMBPAc1-9-reactive CD4+ T cells isolated from naïve B10.PL VB8.2 TCRtransgenic mice in response to stimulation with MBPAc1-9 alone orMBPAc1-9 and graded concentrations of ATRA (0.15, 1.2, 18.8 μg/ml).

FIG. 11 shows a graph depicting the level of IL-2 secreted by Type I NKTcells (Hy1.2) cultured in vitro with graded concentrations of the lipidantigen, αGalCer, and 0, 5, or 10 μg/ml ATRA for 24 hr.

FIG. 12 shows a graph depicting proliferation (as measured by[³H]-thymidine incorporation) of freshly isolated type I NKT spleniccells cultured in the presence of αGalCer and graded concentrations ofATRA, an agonist of RARα (AM580), an agonist of RAR β 2 (AC55649) or anagonist of RARγ (CD1530).

FIG. 13 shows a graph depicting proliferation (as measured by[3H]-thymidine incorporation) of freshly isolated type I NKT spleniccells cultured in the presence of αGalCer and graded concentrations ofan agonist of PPAR-γ (rosiglitazone) or an antagonist of PPAR-γ(GW9662).

FIG. 14 shows a graph depicting proliferation (as measured by[³H]-thymidine incorporation) of freshly isolated type 1 NKT spleniccells cultured in the presence of αGalCer and graded concentrations ofATRA, different retinoic acid analogs, Retinol or Vitamin A.

FIG. 15 shows bar graphs depicting the proliferation (as measured by[³H]-thymidine incorporation) of freshly isolated type I NKT spleniccells (left) or type I NKT cell hybridoma (1.2 Hyb) cultured in thepresence of optimal concentrations of αGalCer (10 ng/ml) and optimalconcentrations of ATRA, Tretinoin, an agonist of RARα (AM580), anagonist of RAR β 2 (AC55649) or an agonist of RARγ (CD1530). The graphsare representative of the data of 3 independent experiments.

FIG. 16 shows graphs depicting the results of in vitro proliferation offreshly isolated splenocytes (top panel) or cytokine release assays oftype 1 NKT cell hybridoma (1.2 Hyb) (bottom panel) cultured with optimalconcentrations of αGalCer (10 ng/ml) in the presence of increasingconcentrations of ATRA or the selective RARγ agonist Tazarotene.Tazarotene shows better inhibition of type I NKT cells than ATRA.

FIG. 17 shows a graph depicting the results of a ex vivo inproliferation assay of type I NKT cells isolated from mice treated withTazarotene, ATRA or DMSO (control) and stimulated with titratedconcentrations of αGalCer.

FIG. 18 shows the analysis of mononuclear cells isolated from livers ofmice injected with ATRA, Tazarotene or DMSO (control) and stained withαGalCer/CD1d-tetramers and pan-anti-TCRβ chain antibodies byflowcytometry. Type I NKT cells are indicated a box. There was nosignificant difference in the numbers (shown next to the box) of type INKT cells in control vs. ATRA or Tazarotene administered animals.

DETAILED DESCRIPTION

The liver harbors a number of specialized cells of the innate immunesystem, including Kupffer cells, Natural Killer (NK) cells, NaturalKiller T (NKT) cells and dendritic cells. NKT cells are unique in thatthey share the cell surface receptors of NK cells (e.g., NK1.1) and inaddition express T cell receptors (TCR), enabling them to recognizelipid antigens in the context of CD1d molecules and bridge the innateimmune responses to adaptive immunity. NKT cells have the ability toregulate the activity of other cells that contribute to inflammation oftissue and the associated cellular damage. Upon activation, NKT cellsrapidly secrete large quantities IFN-γ, IL-4, granulocyte-macrophagecolony-stimulating factor, as well as multiple other cytokines andchemokines. Since NKT cells are capable of secreting both Th1 and Th2cytokines, it is difficult to predict the consequences of NKT cellactivation in vivo. Depending upon context, NKT cell activation triggerscascades of events that promote or suppress different immune responses.In some contexts, activation of NKT cells leads to the activation of NKcells, dendritic cells (DCs) and B cells.

NKT cells recognize lipid antigens presented in the context of themonomorphic MHC class I-like molecule, CD1d. CD1d-restricted NKT cellsare categorized into type I and type II, which recognize different lipidantigens presented by CD1d molecules. While both NKT cell subsets arepredominantly NK1.1+ (mouse) or CD161+/CD56+ (human), their relativenumbers are different in mice and humans: thus, while type I NKT cellspredominate in mice, the type II NKT cell subset predominates in humans.

Type I, also known as invariant NKT cells, express a semi-invariant Tcell receptor (TCR) characterized in mice by Vα14-Jα18 and V08.2, Vβ7,or Vβ2 or in humans by Vα24-JαQ and Vβ11, are strongly reactive with themarine sponge-derived glycolipid α-galactosyl ceramide (αGalCer), andare identified by αGalCer/CD1d-tetramers in flow cytometry. Type I NKTcells also recognize lipid-based antigens, such as, bacterial-derivedlipids and a self-glycolipid, isoglobotrihexosyl ceramide (iGb3). Type INKT cells display memory markers and are unique in storing preformedmRNA for cytokines. Mice lacking the Jα18 gene (Jα18 mice) are deficientonly in type I NKT cells.

Type II NKT cells, which are distinct from type I NKT cells, areregulatory cells that can modulate the activity of several other cellsubsets, including type I NKT cells. Type II NKT cells recognize theself-glycolipid, sulfatide (3′-sulfogalactosyl ceramide) in both miceand in humans. A major subset of type II NKT cells, which can beidentified using sulfatide/CD1d-tetramers in flow cytometry,predominantly utilize the Vβ8.1/Vβ3.1-Jβ2.7 and Val/Vα3-Jα7 genesegments and are reactive to sulfatides. Activation of type II NKT cellscan be evaluated by assessing the in vitro proliferative response oftype II NKT cells to a candidate agent, as well as by assessing C069expression and cytokine secretion profile by intracellular cytokinestaining or real-time PCR for IFN-γ, IL-4 or IL-13. In addition, theability of activated type II NKT cells to anergize type I NKT cells canbe evaluated by assessing the proliferative response of type I NKT cellsto αGalCer (a potent activator of type I NKT cells) using CF8E-dilutionanalysis and intracellular cytokine staining of αGalCer/CO 1 d-tetramercells.

Type I and Type II NKT Cell Activity in the Liver Following of IschemicReperfusion Injury and Concanavalin A-Induced Hepatitis

Reperfusion injury occurs when blood supply is restored to an organ ortissue after a period of ischemia. Hepatic reperfusion injury generallyoccurs in connection with surgery or trauma and plays a major role inthe quality and function of graft tissue after liver transplant.Development of ischemic reperfusion injury (IRI) occurs in at least twophases: an initial period (following from about 1 to about 6 hours ofreperfusion), which is dominated by Kupffer cell activation, release ofreactive oxygen species, CD4⁺ cell recruitment and secretion ofproinflammatory cytokines, and a later period (following the initialphase from about 6 to about 48 hours of reperfusion), which ischaracterized by neutrophil accumulation and induction of necrosis. Asshown in FIG. 2 a , in wild type (WT) mice, a Myeloid (CD11b⁺) cellsubset, CD11b⁺Gr-1^(int), which comprise myeloid precursor cells andmonocytes, are recruited into reperfused tissues at in the early phaseof ischemia and reperfusion injury. The recruitment of myeloid cellsother than neutrophils, such as the CD11b⁺Gr-1^(int) subset, suggeststhat injury is enhanced by recruitment of inflammatory monocytes intoreperfused tissue.

Type I NKT cells also become activated and secrete IFN-γ followingischemic reperfusion. See FIG. 2 c . The role of type I NKT cells in IRIwas evaluated by comparing the livers of wild type (WT) and Jα18^(−/−)mice, which lack type I NKT cells but have normal levels of type II NKTcells. As shown in FIG. 4 , following 90 min of ischemia and 24 hrs ofreperfusion, the cephalad liver lobes of WT mice had large necroticareas whereas necrotic areas in type I NKT cell deficient mice(Jα18^(−/−) mice) were remarkably reduced by comparison. This indicatesa pathogenic role for type I NKT cells in mediating hepatic ischemia andreperfusion injury.

The Effect of Sulfatide-Activated NKT-2 Cells on NKT-1 Cells

As described in U.S. Pat. No. 8,044,029 and U.S. patent application Ser.No. 12/938,315, which are hereby incorporated by reference in theirentirety, administration of a self-glycolipid ligand, sulfatide, as wellas synthetic sulfatide analogs modulate the activity of type I and typeII NKT cells. CD1d-dependent recognition of sulfatide activates type IINKT cells and predominantly plasmacytoid dendritic cells (pDC), but notconventional dendritic cell (cDC) populations (which are normallyactivated by type 1 NKT cells), leading to a rapid recruitment of type INKT cells into liver in an IL-12 and MIP2-dependent fashion. However,the recruited type I NKT cells are not activated, do not secretecytokines and become anergic.

Cellular events involved in the immunoregulatory mechanisms followingsulfatide administration in vivo are shown in FIG. 1 . Administration ofsulfatide a) suppresses in vitro proliferation of type I NKT cells inresponse to stimulation by αGalCer (FIG. 1 b ) and b) increases thepercentage of sulfatide/CD1d-tetramer+ cells with intracytoplasmic IFN-γstaining (FIG. 1 c ).

Secretion of IFN-γ by hepatic type I NKT cells during the early phase ofischemia and reperfusion injury is significantly decreased in micereceiving sulfatide compared to untreated animals. See FIG. 2 c .Further, as with the absence of type I NKT cells in Jα18^(−/−) mice,liver injury following ischemia/reperfusion is significantly reduced inmice treated with sulfatide. See FIG. 4 . This indicates a pathogenicrole for type I NKT cells in mediating hepatic ischemia and reperfusioninjury. The role of IFN-γ secretion by type I NKT cells may be a commonfeature of ischemic organ injury, as kidney ischemia and reperfusioninjury is also attenuated in Jα18^(−/−) mice, which lack type 1 NKT.

Similarly to IRI, sulfatide administration protects against ConcanavalinA (ConA)-induced hepatitis as indicated by reduced hepatocellularnecrosis, see FIG. 1 d , and reduced serum levels of alanineaminotransferase (ALT) and aspartate amino transferase (AST), markers ofhepatocellular damage, see FIG. 1 e . This indicates a pathogenic rolefor type I NKT cells in ConA-induced hepatitis.

Collectively, data from both models of liver damage, IRI andConA-induced hepatitis, are consistent with an immunoregulatory pathwayin which type I NKT cells play a detrimental role and sulfatide-reactivetype II NKT cells play a protective role in hepatic inflammation arisingfrom diverse causes. Such protection is associated with the inhibitionof cytokine secretion by type I NKT cells (inhibited phenotype) and asignificant reduction in hepatic recruitment of immature myeloid cells(CD11b+Gr-1+) CD11b+Gr-1− and NK cells. Inhibition in type I NKT cellsis also associated with the tolerization or modification of conventionaldendritic cells (cDCs) and they together with inhibited type I NKT cellsinhibit activation/expansion of adaptive Th1/Th17 CD4+/CD8+ T cells.This leads to a model where administration of sulfatide stimulates theactivity of type II NKT cells, which in turn inhibit type I NKT cellactivity and the liver damage caused by type I NKT cell-mediatedinflammation. See FIG. 3 .

Role of NKT Cells in Alcoholic Liver Disease

Alcoholic Liver Disease (ALD) develops as a result of damage to livercells caused by excess alcohol consumption. Multiple insults may berequired before clinical manifestations of ALD are observed. However, asdescribed herein, significantly increased numbers of type I NKT cells(αGalCer/CD1d-tetramer+ cells). CD4+ cells, and CD11b+Gr-1+ myeloidcells, but not type II NKT cells or CD11b+Gr-1− cells, are observed inthe liver following chronic alcohol consumption. See Example 6 and FIG.5 . Enhanced expression of CD69 marker further indicates that type I NKTcells are partially activated. Thus, even in the absence of any clinicalsigns of disease (preclinical phase) cells mediating an inflammatoryresponse accumulate in the liver following excess alcohol consumption.In the absence of type I NKT cells (Jα18−/− mice), accumulation ofCD11b+Gr-1+ myeloid cells in the liver following chronic alcoholconsumption is significantly reduced. See Example 6 and FIG. 5 . Thesedata suggest that type I NKT cells are involved in mediating thepreclinical phase of ALD.

Consistent with the data suggesting a role for type I NKT cells in thepreclinical phase of ALD, clinical manifestations of liver damagefollowing excessive alcohol consumption are significantly reduced, asmeasured, for example, by histology and liver enzyme analysis, in type INKT cell deficient (Jα18−/−) mice. See Example 6 and FIGS. 6 and 7 .These data suggest that type I NKT cells play an important role inalcoholic damage to the liver.

NKT cells express T cell receptors, which enable them to recognize lipidantigens in the context of CD1d molecules. Not wishing to be bound by aparticular theory, type I NKT cells are activated in the liver followingethanol consumption by local presentation of oxidized self-lipids byCD1d-expressing antigen presenting cells (APCs). Activated type I NKTcells then initiate a multistep process resulting in Kupffer cellactivation, recruitment/activation of granulocytes followed byinflammatory damage to hepatocytes.

The interplay among NKT cell subtypes following alcohol consumption setsthe stage for liver damage and in severe cases, alcoholic liver disease(ALD). Several embodiments described herein relate to methods andcompositions for manipulating the opposing roles of type I NKT cells andtype II NKT cells and their interactions with other liver cells in orderto treat, alleviate or prevent injury to the liver following alcoholconsumption.

Several embodiments described herein relate to modulating the activityof NKT cell subtypes in order to treat, mitigate or preventalcohol-induced injury to the liver. As described herein, administrationof sulfatide or retinoic acid receptor (RAR) agonists inhibitsalcohol-induced liver damage. For example, injections of sulfatide orretinoic acid receptor (RAR) agonist, All-trans Retinoic Acid (ATRA),during chronic alcohol consumption and prior to ethanol binge had aprotective effect as shown by histological examination and analysis ofliver enzyme levels in serum. See Example 7 and FIGS. 6 and 7 .

Several embodiments relate to a protective role in alcohol induced liverinjury for a major subset of type II NKT cells, which are reactive tosulfatide. Activation of this NKT cell subset by sulfatide inhibits typeI NKT cell-mediated injury following excess alcohol consumption.Sulfatide-mediated protection is associated with activation of type IINKT cells and inhibition of IFN-γ secretion by hepatic type I NKT cellsand suppression of type I NKT cell-mediated recruitment of myeloidcells, for example, the CD11b⁺Gr-1^(int) and Gr-1⁻ subsets, and NK cellsinto the liver.

Sulfatide treatment results in almost complete protection from liverdamage following excess alcohol consumption. See FIGS. 6 and 7 . Notwishing to be bound by a particular theory, the protective effect ofsulfatide is mediated through the direct activation of Type II NKTcells, which exert an inhibitory effect on type I NKT cells and cDCs.

T cell lines from peripheral blood lymphocytes (PBL) of two healthydonors were generated following in vitro stimulation with either αGalCeror sulfatide and analyzed by flow cytometry. Around 2% of the T cellsstain with sulfatide/hCD1d-tetramers in this cell line following twocycles of stimulation. Similar to the murine system, these cells do notuse the invariant Vα24-Vα11 TCR. These data show the presence ofsulfatide-reactive type II NKT cells in healthy individuals and indicatethe effectiveness of using sulfatide/humanCD1d-tetramers in thecharacterization of human type II NKT cells in alcoholic liver disease.

In some embodiments, the sulfatide can be, for example, bovinebrain-derived sulfatide, which is a mixture of about 20 differentspecies obtained from Sigma Inc. (Chicago, Ill., USA). In otherembodiments, the sulfatide is semisynthetic and is a single species ofsulfatide, for example, cis-tetracosenoyl sulfatide or lysosulfatideobtained from Maitreya Inc, (Pleasant Gap, Pa., USA). In still otherembodiments, the sulfatide can be a totally synthetic sulfatide.

Sulfatide derived from bovine brain myelin is comprised of a 2:1 mixtureof saturated/unsaturated major acyl chains (C₂₄), with unsaturationoccurring at C₁₉. Several embodiments relate to the use of syntheticsulfatide analogs, such as analogs with saturated acyl chain (C₂₄) andunsaturated chains (C₂₄: 1) as well as analogs comprised of differentlengths of acyl chains in the fatty acid or sphingosine moiety (shorteras well as longer, for example, C₁₈, C₃₂) and positional isomers with 3′vs. 4′-sulfated group on the galactose moiety (3′-803 vs. 4′-803).

In some embodiments the sulfatide has the following chemical formula I:

wherein R₁ can be a bond, a hydrogen, a C₁ to C₃₀ alkyl, a C₁ to C₃₀substituted alkyl, a C₁ to C₃₀ alkenyl, a C₁ to C₃₀ substituted alkenylor a C₅ to C₁₂ sugar; R₂ can be a hydrogen, a hydroxy group, a methoxygroup, or an alkoxy group; R₃ can be a hydrogen, a hydroxy group, amethoxy group, an ethoxy group, or an alkoxy group; R₄ can be ahydrogen, a hydroxy group or an alkoxy group; R₅ can be a hydrogen, ahydroxy group, a carbonyl, an alkoxy group or a bond; R₆ can be a C₁ toC₄₀ alkyl, a C₁ to C₄₀ substituted alkyl, a C₁ to C₄₀ alkenyl, a C₁ toC₄₀ substituted alkenyl, or a C₁ to C₄₀ alkynl; R₇ can be a C₁ to C₄₀alkyl, a C₁ to C₄₀ substituted alkyl, a C₁ to C₄₀ alkenyl, a C₁ to C₄₀substituted alkenyl, or a C₁ to C₄₀ alkynl; and R₈ can be a hydrogen, ahydroxy group, a carbonyl, an alkoxy group or a bond.

In other embodiments, the sulfatide has the following chemical formulaII:

wherein R₁ is selected from the group consisting of a C₁ to C₄₀ alkyl, aC₁ to C₄₀ substituted alkyl, a C₁ to C₄₀ alkenyl, a C₁ to C₄₀substituted alkenyl and a C₁ to C₄₀ alkynl; and R₂ is selected from thegroup consisting of a hydrogen, a hydroxyl group, a carbonyl, an alkoxygroup and a bond.

In another embodiment, the sulfatide has the following chemicalstructure:

As used herein, the term “alkyl” means any unbranched or branched,saturated hydrocarbon. The term “substituted alkyl” means any unbranchedor branched, substituted saturated hydrocarbon. Cyclic compounds, bothcyclic hydrocarbons and cyclic compounds having heteroatoms, are withinthe meaning of “alkyl.”

As used herein, the term “substituted” means any substitution of ahydrogen atom with a functional group.

As used herein, the term “functional group” has its common definition,and refers to chemical moieties preferably selected from the groupconsisting of a halogen atom, C₁-C₂₀ alkyl, substituted C₁-C₂₀ alkyl,perhalogenated alkyl, cyloalkyl, substituted cycloalkyl, aryl,substituted aryl, benzyl, heteroaryl, substituted heteroaryl, cyano, andnitro.

As used herein, the terms “halogen” and “halogen atom” refer to any oneof the radio-stable atoms of column 17 of the Periodic Table of theElements, preferably fluorine, chlorine, bromine, or iodine, withfluorine and chlorine being particularly preferred.

As used herein, the term “alkenyl” means any unbranched or branched,substituted or unsubstituted, unsaturated hydrocarbon. The term“substituted alkenyl” means any unbranched or branched, substitutedunsaturated hydrocarbon, substituted with one or more functional groups,with unbranched C₂-C₆ alkenyl secondary amines, substituted C₂-C₆secondary alkenyl amines, and unbranched C₂-C₆ alkenyl tertiary aminesbeing within the definition of “substituted alkyl.” Cyclic compounds,both unsaturated cyclic hydrocarbons and cyclic compounds havingheteroatoms, are within the meaning of “alkenyl.”

As used herein, the term “alkoxy” refers to any unbranched, or branched,substituted or unsubstituted, saturated or unsaturated ether.

As used herein, the term “sulfatide” retains its general accustomedmeaning and refers to a cerebroside sulfuric ester containing one ormore sulfate groups in the sugar portion of the molecule.

As used herein, the term “cerebroside” refers to any lipid compoundcontaining a sugar, and generally of the type normally found in thebrain and nerve tissue.

The compounds of formula (I), (II) and (III) may be in the form ofpharmaceutically acceptable nontoxic salts thereof. Salts of formula(I), (II) and (III) include acid added salts, such as salts withinorganic acids (e.g., hydrochloric acid, sulfuric acid, nitric acid andphosphoric acid) or with organic acids (e.g., acetic acid, propionicacid, maleic acid, oleic acid, palmitic acid, citric acid, succinicacid, tartaric acid, fumaric acid, glutamic acid, pantothenic acid,laurylsulfonic acid, methanesulfonic acid and phthalic acid).

The compounds of formula (I), (II) and (III) may be in the form ofsolvates thereof (e.g., hydrates).

The compounds of formula (I), (II) and (III) can be produced by anypurposive method to synthesize sulfatides.

The compounds of formulas (I), (II) and (III) can also be isolated fromnatural products (e.g., biological organisms) and purified by columnchromatography or the like.

In one embodiment, the sulfatide has the chemical formula: (2S, 3R,4E)-N-nervonic-1-[-D-(3-sulfate)-galactopyranosyl]-2-amino-octadecene-3-ol.This chemical formula is also referred to as cis-tetracosenoylsulfatide.

In some embodiments, the specific amount of sulfatide administered to apatient will vary depending upon the disease or condition being treated,as well as the age, weight and sex of the patient being treated.Generally, to achieve such a final concentration in, e.g., theintestines or blood, the amount of sulfatide molecule in a single dosagecomposition of the present embodiments will generally be about 0.1milligrams to about 100 milligrams, preferably about 2.0 milligrams toabout 60 milligrams, more preferably about 20 milligrams to about 50milligrams. Likewise, the amount of a secondary therapeutic compound ina single oral dosage composition of the present embodiments willgenerally be in the range of about 0.01 milligrams to about 1000milligrams, more preferably about 0.1 milligrams to about 100milligrams. Obviously, the exact dosage will vary with the disease ordisorder being treated, the preferred ranges being readily determinable.

In one embodiment, 0.1-10 mg/kg body weight of sulfatide areadministered to the patient. More preferably, 1-10 mg/kg body weight ofsulfatide are administered. Preferably, this dosage is repeated each dayas needed.

Effect of Retinoic Acid Receptor (RAR) Agonists on NKT Cell Activity

As described herein, administration of the Retinoic Acid Receptor (RAR)agonist, all trans retinoic acid (ATRA), significantly inhibits liverdamage caused by alcohol consumption. Ethanol ingestion depletes liverretinyl esters and alters physiological levels of all trans retinoicacid (ATRA), a biologically active from of vitamin A that supports manybiological functions and can enhance generation, stability and functionof naïve CD4+ T cell differentiation into FoxP3+ Tregs even in thepresence of IL-6. Several embodiments described herein relate to thefinding that ATRA inhibits type I NKT cell effector function, includingsuppressing cytokine secretion by these cells. See Examples 8, 9 and 11and FIGS. 8, 10, and 11 . Further, ATRA has a direct inhibitory effecton type 1 NKT cell activity, exerting its inhibitory effect in theabsence of antigen-presenting cells. See Example 11, FIG. 11 . Inaddition, ATRA does not directly inhibit the activity of conventionalMHC-restricted CD4+ T cells that recognize protein antigens, such asmyelic basic protein or MBPAc1-9. Not wishing to be bound by aparticular theory, these data indicate that ATRA protects against liverdamage caused by excessive alcohol consumption by inhibiting type I NKTcells.

As described herein, RAR agonists induce inhibition in type I NKT cells.See FIGS. 12, 14 and 15-17 . Further, liver damage caused by type I NKTcell mediated inflammation resulting from excess alcohol consumption canbe prevented, reduced or mitigated by administration of an RAR agonist.The liver can become inflamed for a variety of different reasons. Forexample, liver inflammation can be caused by bacterial or viralinfection, injury, or attack from one's own immune system. Whileinflammation is normally a protective response and a required step ofthe healing process, prolonged or chronic inflammation can cause injury.Several embodiments described herein relate to the RAR agonist mediatedmodulation of the innate immune mechanisms leading to liver injuryfollowing, related to or caused by inflammation. Some embodiments relateto methods and compositions for RAR agonist mediated inhibition of typeI NKT cell activity which modulate interactions among the components ofthe innate immune system to provide tolerance to gut-derived ormetabolite-derived antigens without affecting or minimally affecting theinnate immune response to non-self identified pathogens.

As RAR agonists can directly anergize Type I NKT cells, RAR agonists maybe used to treat any indication in which Type I NKT cells play apathogenic role. Some examples of diseases which can be treated by theembodiments of the present disclosure include, alcohol inducedhepatitis, non-alcoholic steatosis hepatitis, cirrhosis, fulminatingcirrhosis, idiopathic hepatitis, viral-induced hepatitis (A, B, C andother), inflammatory hepatitis associated with hepato-biliary carcinoma,multiple sclerosis, type 1 diabetes, ischemic reperfusion injury, solidorgan transplantation, systemic lupus erythematosus, rheumatoidarthritis, amyotrophic lateral sclerosis, and inflammatory bowel disease(Crohn's and colitis).

In some embodiments, are various indications of autoimmune or immunerelated diseases or disorders are treated, prevented or mitigated by RARagonist mediated inhibition of type I NKT cell activity. In particular,one aspect of the present embodiment is related to a method of treatinga patient suffering from symptoms of an autoimmune or immune relateddisease or disorder, such as, for example, multiple sclerosis, systemiclupus erythematosus, AIDS, Alzheimer's disease, rheumatoid arthritis,insulin dependent diabetes mellitus, autoimmune hepatitis, asthma, andceliac disease with an effective amount of an RAR agonist. In someembodiments, RAR agonist mediated inhibition of type I NKT cell activitytreats, prevents or mitigates asthma symptoms.

Some embodiments relate a method of inhibiting or preventing type I NKTcell mediated inflammation following ischemic reperfusion byadministering an RAR agonist. Type I NKT cells play a pathogenic role inconditions such as ischemia and reperfusion injury. Reperfusion injurycan occur in a variety of tissues when blood supply is restored after aperiod of ischemia. Examples include skeletal muscle tissue following acrush injury, cardiac muscle in connection with a myocardial infarctionor cardiac surgery, or ischemic heart disease, neural tissue inconnection with a stroke or brain trauma, and hepatic and renal tissuein connection with surgery or trauma. Ischemic reperfusion injury alsoplays a major role in the quality and function of graft tissue in organtransplant. Ischemia and reperfusion injury is a major cause forincreased length of hospitalization and decreased long-term graftsurvival. In some embodiments, RAR agonist mediated inhibition of type INKT cell activity inhibits or prevents hepatic ischemic reperfusioninjury in connection with surgery or trauma.

Embodiments described herein relate to the inhibition of type I NKT cellactivity by one or more retinoic acid receptor (RAR) agonists. Retinoicacid receptors comprise three major subtypes: RARα, RAR β, and RARγ.Some embodiments relate to the inhibition of type I NKT cell activity byone or more pan-active RAR agonists, precursors of such pan-active RARagonists and mixtures thereof. As used herein, the term “pan-active RARagonist” refers to a RAR agonist which affects, for example, activates,RARα, RARβ, and RARγ substantially equally or non-selectively. Someembodiments relate to the inhibition of type I NKT cell activity by oneor more active RAR agonists effective to selectively, or evenspecifically, affect, for example, activate, at least one, andpreferably both, of RARβ and RARγ relative to RARα, precursors of suchactive RAR agonists and mixtures thereof. As used in this context, theterm “selectively” means that the RAR agonist precursors of the RARagonist and mixtures thereof are more effective, preferably at leastabout 10 or about 100 times to about 1000 times or more as effective, toaffect at least one, and preferably both, of RAR β and RARγ relative toRARα. Some embodiments relate to the inhibition of type 1 NKT cellactivity by one or more subtype-selective RAR agonists, precursors ofsuch subtype-selective RAR agonists and mixtures thereof. As usedherein, the term “subtype-selective RAR agonist” refers to a RAR agonistwhich selectively affects, for example, activates one RAR subtype.Retinoid compounds having RARα, RAR β, and RARγ-selectivity are known inthe art and disclosed, for example, in U.S. Pat. Nos. 6,534,544 and6,025,388 which are herein incorporated by reference in their entirety.

Several embodiments relate to a method of inhibiting pro-inflammatorytype I NKT cell activity by administering one or more retinoic acidreceptor (RAR) agonists. Examples of RAR agonists include, but are notlimited to, the RAR agonists listed in Table 1.

TABLE 1 RAR agonists Compound Name Specificity Structure TretinoinPan-RAR agonist

9-cis RA Pan-RAR and RXR agonist

13-cis-RA Pan-RAR agonist

Fenretinide RAR agonist RAR-independent effects

EC 23 Pan-RAR agonist

TTNPB Pan-RAR agonist

Ch 55 Pan-RAR agonist

Tazarotene RARβ/γ agonist

BMS 753 RARα agonist

AM80 RARα agonist

AM580 RARα agonist

AC55649 RARβ2 agonist

AC261066 RARβ2 agonist

Adapalene RARβ and γ agonist

CD437 RARγ agonist

CD1530 RARγ agonist

CD2665 RARγ agonist

MM11253 RAR agonist

LE135 RARβ agonist

BMS493 Pan-RAR inverse agonist

BMS453 RARβ agonist RARα or RARγ Antagonist

In some embodiments, pro-inflammatory type I NKT cell activity isinhibited by one or more RAR agonists selected from the group consistingof ATRA, retinol, 9-cis-RA or 13-cis-RA, tretinoin, AM580, AC55649,CD1530 or Tazarotene. In some embodiments, pro-inflammatory type I NKTcell activity is inhibited by one or more polyolefinic retinoids, suchas isoretinoin and acitretin. In some embodiments, pro-inflammatory typeI NKT cell activity is inhibited by one or more RAR agonists selectedfrom the group consisting of etretinate, acitretin and isotretinoin.

Several embodiments relate to the inhibition of pro-inflammatory type INKT cell activity by tazarotene, tazarotenic acid or a mixture thereof.Tazarotene is an ethyl ester prodrug that is metabolized to thecorresponding free acid, tazarotenic acid. Tazarotene has a rigidring-locked structure that offers limited conformational flexibilitycompared to all-trans-retinoic acid, the natural ligand for the retinoicacid receptors (RARs). This structural change confers tazarotenic acidwith specificity for the RARs and selectivity for RARβ and RARγ.

Examples of RAR agonists further include esters of cis- andtrans-retinoic acids, for example, alkyl esters, such as primary,secondary or tertiary alcohols, including but not limited to: methyl,ethyl, propyl, iso-propyl, butyl, iso-butyl, hexyl, heptyl, ethylhexyl,octyl, nonyl, lauryl, oleyl, stearyl, hydroxyethyl, hydroxypropyl,benzyl, alpha-methylbenzyl, alpha-propylphenyl, amyl, iso-amyl, anisyl,cetyl, menthyl, cinnamyl, pinacol, furyl, or myristyl.

Pharmaceutically acceptable salts of RAR agonists can also be used toinhibit type I NKT cell activity. Compounds disclosed herein whichpossess a sufficiently acidic, a sufficiently basic, or both functionalgroups, and accordingly can react with any of a number of organic orinorganic bases, and inorganic and organic acids, to form a salt.

Examples of acids that may be used to form acid addition salts from RARagonists with basic groups include inorganic acids such as hydrochloricacid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid,and the like, and organic acids such as p-toluenesulfonic acid,methanesulfonic acid, oxalic acid, p-bromophenyl-sulfonic acid, carbonicacid, succinic acid, citric acid, benzoic acid, acetic acid, and thelike. Examples of such salts include the sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, acetate, propionate, decanoate, caprylate, acrylate, formate,isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate,succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate,xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate,citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate,methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate, mandelate, and the like.

Examples of bases that may be used to form base addition salts from RARagonists with acidic groups include, but am not limited to, hydroxidesof alkali metals such as sodium, potassium, and lithium; hydroxides ofalkaline earth metal such as calcium and magnesium; hydroxides of othermetals, such as aluminum and zinc; ammonia, and organic amines, such asunsubstituted or hydroxy-substituted mono-, di-, or trialkylamines;dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine;diethylamine; triethylamine; mono-, bis-, or tris-(2-hydroxy-lower alkylamines), such as mono-, bis-, or tris-(2-hydroxyethyl)amine,2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine,N,N-di-lower alkyl-N-(hydroxy lower alkyl)-amines, such asN,N-dimethyl-N-(2-hydroxyethyl)amine, or tri-(2-hydroxyethyl)amine;N-methyl-D-glucamine; and amino acids such as arginine, lysine, and thelike.

As used herein, the term “patient” refers to the recipient of atherapeutic treatment and includes all organisms within the kingdomanimalia. In preferred embodiments, the animal is within the family ofmammals, such as humans, bovine, ovine, porcine, feline, buffalo,canine, goat, equine, donkey, deer and primates. The most preferredanimal is human.

As used herein, the terms “treat” “treating” and “treatment” include“prevent” “preventing” and “prevention” respectively.

As used herein the term “an effective amount” of an agent is the amountsufficient to treat, inhibit, or prevent liver damage resulting frompro-inflammatory type 1 NKT cell activity.

Some embodiments relate to pretreatment of patients with a sulfatideand/or an RAR agonist prior to clinical manifestation of ALD. In severalembodiments, patients are treated with an effective amount of sulfatideand/or an RAR agonist prior to binge alcohol consumption. In some otherembodiments, patients are treated with an effective amount of sulfatideand/or an RAR agonist from about 0.5 hour to about 18 hours prior tobinge alcohol consumption. In some other embodiments, patients aretreated with an effective amount of sulfatide and/or an RAR agonistduring a period of chronic alcohol consumption.

In some embodiments, the specific amount of RAR agonist administered toa patient will vary depending upon the disease or condition beingtreated, as well as the age, weight and sex of the patient beingtreated. Generally, to achieve such a final concentration in, e.g., theintestines or blood, the amount of RAR agonist in a single dosagecomposition of the present embodiments will generally be about 0.1milligrams to about 100 milligrams, preferably about 2.0 milligrams toabout 60 milligrams, more preferably about 20 milligrams to about 50milligrams. Examples of suitable doses include: between about 0.1 toabout 10 mg/day; between about 0.5 to about 2 mg/day; between about0.01-100 mg/day; between about 1 to about 50 mg/day; between about 0.1mg/day to about 1.0 mg/day; between about 1.0 mg/day and about 5.0mg/day; between about 5.0 mg/day and about 10.0 mg/day; between about10.0 mg/day and about 15 mg/day; between about 15.0 mg/day and about20.0 mg/day; between about 20.0 mg/day and about 25.0 mg/day; betweenabout 30.0 mg/day and about 35.0 mg/day; between about 35.0 mg/day andabout 40.0 mg/day; between about 40.0 mg/day and about 45.0 mg/day;between about 45.0 mg/day and about 50.0 mg/day; between about 50.0mg/day and about 55.0 mg/day; between about 55.0 and about 60.0 mg/day;between about 60.0 mg/day and about 65.0 mg/day; between about 65.0mg/day and about 70.0 mg/day; between about 70.0 mg/day and about 75.0mg/day; between about 75.0 mg/day and about 80.0 mg/day; between about80.0 mg/day and about 85.0 mg/day; between about 85.0 and about 90.0mg/day; between about 85.0 mg/day and about 90.0 mg/day; between about90.0 mg/day and about 95.0 mg/day; and between about 95.0 mg/day andabout 100.0 mg/day. Obviously, the exact dosage will vary with thedisease or disorder being treated, the preferred ranges being readilydeterminable.

When tazarotene is orally administered to effect a reduction in type INKT cell activity, the daily dosage of tazarotene preferably is in arange of about 0.3 mg/day to about 7 mg/day or about 8 mg/day, morepreferably in a range of about 0.6 mg/day to about 6.5 mg/day or about 7mg/day. In some embodiments, orally administered tazarotene isadministered in daily dosages of tazarotene including 0.4 mg/day, 0.75mg/day, 1.5 mg/day, 2.8 mg/day, 3 mg/day, 4.5 mg/day, 6 mg/day and 6.3mg/day.

In accordance with the embodiments, RAR agonist can be administered toalleviate a patient's symptoms, or can be administered to counteract amechanism of the disorder itself. In certain embodiments, RAR agonistmay be administered as a prophylactic measure. In some embodimentsmultiple doses of RAR agonist is administered. It will be appreciated bythose of skill in the art that these treatment purposes are oftenrelated and that treatments can be tailored for particular patientsbased on various factors. These factors can include the age, gender, orhealth of the patient, and the progression of autoimmune or immunerelated disease or disorder. The treatment methodology for a patient canbe tailored accordingly for dosage, timing of administration, route ofadministration, and by concurrent or sequential administration of othertherapies.

In some embodiments, one or more RAR agonist compounds can beadministered alone or in combination with another therapeutic compound.For example, one or more RAR agonist compounds can be administered incombination with a sulfatide. Any currently known therapeutic compoundused in treatment of the alcoholic liver disease, inflammatory disease,autoimmune disease or reperfusion injury can be used. In someembodiments, RAR agonist can be administered in combination withhydrogen sulfide (H₂S). In some embodiments RAR agonist can beadministered in combination with antioxidants. In some embodiments RARagonist can be administered in combination with, for example,corticosteroids, biologics (e.g. anti-TNF-alpha and anti-IL-6),immunomodulators (e.g. RU-486), disease modifying anti-rheumatic drugs(DMARDS, such as leflunomide), COX-2 inhibitors (celecoxib),non-steroidal anti-inflammatory drugs (NSAIDS, such as naproxen), oralanti-diabetic (OAD, such as metformin or sitaglipten), GLP-1 agonists,insulin, PPAR agonists/antagonists, EGF mediators (anti-cancer agents),other agents effective to treat hepatic cancers, cell-based therapiesfor liver cancers; interferons (IFN) for Hepatitis C, multiple sclerosisor lupus erythematosus; and LFA-1 antagonists.

The RAR agonist compounds and sulfatide compounds described herein maybe used as an active ingredient incorporated into a pharmaceuticalcomposition. In some embodiments the pharmaceutical composition maycomprise a single active ingredient. In some embodiments thepharmaceutical composition may comprise two, three, four, five or moreactive ingredients. All modes of administration are contemplated, forexample, orally, rectally, parenterally, topically, or by intravenous,intramuscular, intrasternal or subcutaneous injection or in a formsuitable by inhalation. The formulations may, where appropriate, beconveniently presented in discrete dosage units and may be prepared byany of the methods well known in the art of pharmacy. The activeingredients will ordinarily be formulated with one or morepharmaceutically acceptable excipients in accordance with known andestablished practice. Thus, the pharmaceutical composition can beformulated as a liquid, powder, elixir, injectable solution, suspension,suppository, etc.

Active ingredients according to some embodiments can be formulated foradministration for oral administration as tablets, hard gelatincapsules, soft gelatin capsules, comprising the active in the form of apowder, a blend with excipients, a liquid or solution, a suspension, asolid solution. Active ingredients according to some embodiments can beformulated for administration for intra-oral administration (sublingualor buccal) as a solid dosage form rapidly dissolving or effervescenttablets, thin films, buccal wafers, or as a liquid or semi-solid form,such as a gel, solution, emulsion, suspension. Active ingredientsaccording to some embodiments can be formulated for administration forinjection as an aqueous or non-aqueous solution, suspension or emulsion.Oil-based solutions and suspensions comprise mixtures of natural and orsynthetic oils such as soybean oil, cotton seed oil, mineral oil, sesameoil, castor oil, hydrogenated vegetable oils, beeswax. Activeingredients according to some embodiments can be formulated foradministration for transdermal administration as a cream, a gel, anemulsion, an aqueous-based solution, an oil-based solution, asuspension, a film, a patch, a foam. Active ingredients according tosome embodiments can be formulated for administration for intranasaladministration as a powder, suspension, solution, emulsion. Activeingredients according to some embodiments can be formulated foradministration for pulmonary delivery as a micronized powder. Oraladministration is associated with first pass metabolism as well asinduction of metabolizing enzymes. Thus dosage strength and dosingregimen of oral administered retinoic acids may be tailored for optimaleffect. Alternative routes of delivery, e.g. sublingual, buccal,injection, pulmonary and transdermal, may be a preferred over oraladministration. Alternative routes of administration, such as those asdescribed, avoid first pass metabolism and GI absorption, demonstrateless enzyme induction and provide steady repeat dose pharmacokinetics.

Pharmaceutical formulations according to the present embodiments may beimmediate release or modified release (e.g., sustained release,pulsatile release, controlled release, delayed release, slow release).Because it is immediately active, pharmacological amounts of orallyadministered Retinoid isomers may have side effects. These side effectshave been a serious limitation to the use of oral retinoids in therapy.Although topically applied retinoids carry little teratogenic liabilitythere are other toxicities associated with this route of administrationthat limit their use including skin irritation. A major reason for bothoral and topical toxicity is that the retinoids are totally andimmediately available upon administration. A process whereby a retinoidcan be made available in vivo more slowly and more continuously wouldavoid peaks and valleys in the availability of the retinoid therebyproviding an effective in vivo level of the compound over a moreprolonged period of time and also avoiding or substantially reducing thetoxicities that often result from the sudden availability of excessiveamounts of the substance. An oil based injectable formulation ofretinol, ester of retinol, and in particular a fatty ester of retinol,retinoic acid or a retinoic acid ester could be administeredintra-muscularly on a weekly basis and provide a systemic slow-releasedelivery, according to such principles.

In some embodiments, preparation of all-trans-retinoic acid tert-butylester is as follows. To a solution of all-trans retinoic acid (100 mg,0.33 mmol) in anhydrous ether was added oxalyl chloride (42.3 mg, 0.333mmol) at 0.degree. C. and stirred at that temperature for 30 minutes andpyridine (28.7 mg, 0.363 mmol), 2-methyl-2-propanol (26.8 mg, 0.363mmol) was added and stirred at room temperature in dark after which timethe reaction was complete as indicated by the TLC. The reaction mixturewas then quenched with water and extracted with ether (3.times.10 ml),saturated sodium bicarbonate solution (3.times.5 ml) and again withwater (3.times.5 ml), dried (MgSO.sub.4) and evaporated. The thickresidue was redissolved in hexane and applied on silica Sep-Pakcartridge (2 g). Elution with hexane/ethyl acetate (9.7:0.3) providedthe butyl ester of retinoic acid. Final purification was achieved byHPLC (10 mm.times.25 cm Zorbax-Sil column, 4 mL/min) usinghexane/isopropanol (90:10) solvent system. Pure all-trans retinoylbutyrate 2 (98 mg, 82.6%) was eluted at as a thick oil.

Some embodiments relate to formulation of butyl-retinoic ester forinjection as described below. 10 g butyl-retinoic ester solution isdissolved in a mixture of 73 g cottonseed oil containing 0.1 g butylatedhydroxyanisole, and 10 g benzyl alcohol at slightly elevated temperatureand with high-shear mixing under aseptic conditions. The mixture issterile filtered and filled into a syringe for later use. To beadministered by intramuscular injection. Some embodiments relate toformulation of retinol palmitate in cottonseed oil as above forintramuscular injection.

Some embodiments relate to formulation of an oral dosage form of one ormore active ingredient of the present embodiments as described below. 10g retinoic acid is dissolved in a mixture of beeswax, butylatedhydroxyanisole, edetate disodium, hydrogenated soybean oil flakes,hydrogenated vegetable oils and soybean oil alcohol at slightly elevatedtemperature and with high-shear mixing. The mixture is sealed intosoft-gelatin capsules at dosage strengths of 2 mg, 5 mg and 10 mg fororal administration.

Some embodiments relate to formulation of a bioadhesive buccal tabletcontaining one or more active ingredient of the present embodiments. Ablend of excipients is prepared containing 24% active ingredient, 21%HPMC, 18% Corn Starch, 24% Lactose, 1% Silica, 2.5% Polycarbophil(Noveon), 7.5% Carbomer 974P, 1.2% Talc and 0.7% Magnesium Stearate. Theblend was pressed into tablets approximately lcm in diameter.

Some embodiments relate to formulation of a sublingual film containingone or more active ingredient of the present embodiments. The followingare mixed in 50 g water: 3 g Methocel E5, 5 g Methocel E50, 1 g Klucel,1 g Maltodextrin, 1 g citric acid, 3 g sucralose, 5 g Orange flavor, 0.2g paraben, 0.1 edetate sodium and 5 g Sorbitol. 1 g retinoic acid isadded and the mixture is degassed with stirring. The composition isthinly spread across a polyester film support and allowed to dry in theair in the absence of any direct light to avoid degradation. The film isthen cut to size to generate doses of 2 to 10 mg.

Some embodiments relate to a kit, which may include one or moresulfatides, RAR agonists or any combination thereof, preferably as apharmaceutical composition. In some embodiments, cis-tetracosenoyl isprovided in a pharmaceutically acceptable carrier. In some embodiments,ATRA is provided in a pharmaceutically acceptable carrier. In someembodiments, tazarotene is provided in a pharmaceutically acceptablecarrier. In several embodiments, hydrogen sulfide (H₂S) may optionallybe provided. In several embodiments, one or more antioxidants mayoptionally be provided. In several embodiments, kits may furthercomprise suitable packaging and/or instructions for use. Kits may alsocomprise a means for the delivery of the one or more sulfatides, RARagonists or any combination thereof, such as an inhaler, spray dispenser(e.g., nasal spray), syringe for injection, needle, IV bag or pressurepack for capsules, tables, suppositories. The one or more sulfatides,RAR agonists or any combination thereof can be in a dry or lyophilizedform or in a solution, particularly a sterile solution. When in a dryform, the kit may comprise a pharmaceutically acceptable diluent forpreparing a liquid formulation. The kit may contain a device foradministration or for dispensing the compositions, including, but notlimited to, syringe, pipette, transdermal patch, or inhalant. Someembodiments relate to kits that contain sufficient dosages of thecompounds or composition to provide effective treatment for anindividual for an extended period, such as a week, 2 weeks, 3 weeks, 4weeks, 6 weeks, or 8 weeks or more.

In one example embodiment, a 70 kg adult patient at risk of chemicalliver damage from prescription drugs or drugs of abuse is given a dailyi.m. injection of 7 mg tazarotene in 1.0 ml phosphate buffered saline totreat liver damage. This dosage can be adjusted based on the results ofthe treatment and the judgment of the attending physician. Treatment ispreferably continued for at least about 1 or 2 weeks, preferably atleast about 1 or 2 months, and may be continued on a chronic basis.

In another example embodiment, a 70 kg adult patient at risk of chemicalliver damage from prescription drugs or drugs of abuse is given an oraldose of an RAR agonist sufficient to inhibit activity of type 1 NKTcells. Liver damage may be monitored by analysis serum liver enzymelevels. The dosage of RAR agonist can be adjusted based on the resultsof the liver function test and the judgment of the attending physician.Treatment is preferably continued for at least about 1 or 2 weeks,preferably at least about 1 or 2 months, and may be continued on achronic basis. In some embodiments the duration of treatment coincideswith duration of behavior placing the patient at risk for chemical liverdamage. In some embodiments, the RAR agonist is selected from the groupconsisting of ATRA and tazarotene. In some embodiments, the patient isfurther administered a dose of sulfatide sufficient to activate type IINKT cells.

While the foregoing written description enables one of ordinary skill tomake and use what is considered presently to be the best mode thereof,those of ordinary skill will understand and appreciate the existence ofvariations, combinations, and equivalents of the specific embodiment,method, and examples herein. The present embodiments should thereforenot be limited by the above described embodiment, method, and examples,but by all embodiments and methods within the scope and spirit of thepresent embodiments.

The following Examples are presented for the purposes of illustrationand should not be construed as limitations.

Example 1

Sulfatide Mediates Inhibition of Type I NKT Cells while ActivatingSulfatide/CD1d-Tetramer+ Cells

A dose of 20 μg sulfatide was injected intraperitoneally in mice.Following sulfatide injection, MNCs from liver were isolated and labeledwith CFSE. The cells were cultured with αGalCer (10 ng/ml) for 96 hoursin the presence or absence of 10 ng/ml cytokine IL-2. Cells werecultured with 10 ng/ml IL-12 alone as a control. Following culture,cells were harvested, stained with anti-TCRβ mAb andαGalCer/CD1d-tetramers, FACs sorted and CFSE dilution analysis wasperformed on αGalCer/CD1d-tetramer⁺ (type I NKT) cells. See FIG. 1A. Asshown in FIG. 1B, after sulfatide administration, type I NKT cells failto proliferate in response to an in vitro challenge with αGalCer.

Liver cells isolated from PBS or 20 μg sulfatide injected mice weresorted into sulfatide/CD1d-tetramer+ and tetramer− populations andstained for intracytoplasmic IFN-γ+. As shown in FIG. 1C,sulfatide-injected mice have the greatest % IFN-γ positive cells.

Administration of sulfatide results in both the inhibition of type I NKTcell proliferation and increase in the % IFN-γ positive cells. Not to bebound by a particular theory, it is suggested that sulfatideadministration activates both pDC and type II NKT cells which secretecytokines and chemokines and this interaction results in cDC-mediatedinhibition induction in type I NKT cells.

Example 2

Sulfatide Administration Protects Against ConA-Induced Hepatitis

Female C57BL/6 mice were treated with 8.5 mg/kg of Concavalin A (ConA)(dissolved in pyrogen free phosphate buffer saline, PBS) intravenously(i.v.), which induces liver damage similar to that caused by hepatitis.Immediately after ConA injection, mice were injected intraperitoneally(i.p.) with 20 μg (1 mg/kg/m) of bovine brain sulfatide or PBS.

Damage to the liver gives rise to telltale abnormalities (suggestingliver disease) detectable by liver function tests. For example, viralhepatitis can cause the alanine amino transferase (ALT) and aspartateamino transferase (AST) enzymes in injured liver cells to spill into theblood stream and increase their level in the blood. To test for liverdamage, serum was collected and levels of serum enzymes, ALT and AST,were measured at 0, 6, 12, 24, 48 and 72 hours following Con A or ConA+sulfatide injection. Serum enzyme levels were measured with the helpof Laboratory Corporation of America, San Diego, Calif. As shown in FIG.1 e , comparable levels of serum enzymes were observed 6 hours after ConA or Con A+sulfatide injection, however, both ALT and AST serum levelswere lower at 12, 24, and 48 hours in mice injected with both ConA andsulfatide. In Con A (●) injected mice, serum ALT and AST peaked around12 h (ALT-15.8×10³ IU/L and AST-22.7×10³ IU/L) and returned to base lineby 48 hours. In contrast, following combination of Con A+sulfatide (◯)injection, a significant decrease in serum level of ALT and AST(ALT≈2.5×10³ IU/L and AST≈5.4×10³ IU/L) by 12 h was recorded andreturned to base line by 24 h. Values are mean±SD of 5 mice per group.P<0.001.

Mice were sacrificed at 12, 24, 48, 72, and 96 hours after treatment andtheir livers were collected. Liver tissue was fixed in 10% formaldehydesolution and kept at room temperature until use. Histologicalexamination using hematoxylin and eosin (H&E) staining was performed atPacific Pathology Inc., San Diego, Calif.

As shown in FIG. 1 d , H&E staining demonstrated markedly improvedhepatic histology in Con A+bovine brain sulfatide treated mice relativeto mice treated with Con A alone. Histological examination showeddiffuse and massive infiltration and severe necrosis at the indicatedtime points following Con A injection mice, FIG. 1 d , top panels. Incontrast, sulfatide+Con A injection was associated with mild injury interms of less infiltration and less necrosis in the 12 h to 48 h liversections and histology returned to normal by 72 h, FIG. 1 d , bottompanels.

Taken together, results obtained in Example 1 and Example 2 indicatethat (a) sulfatide mediates inhibition of type I NKT cells in the liverand (b) sulfatide administration activates sulfatide/CD1d-tetramer+cells and protects against ConA-induced hepatitis. This indicates thattolerized cDCs and anergic type I NKT cells collectively lead to asignificant inhibition of a detrimental inflammatory cascade.

Example 3

Analysis of the Effect of the Immune Response to Hepatic Ischemia andReperfusion Injury on the Composition of the CD11b+Gr-1+ Cell Population

The hepatic ischemia and reperfusion injury model was established asdescribed in Shen X D, Ke B, Zhai Y, et al., CD154-CD40 T-cellcostimulation pathway is required in the mechanism of hepaticischemia/reperfusion injury, and its blockade facilitates and depends onheme oxygenase-1 mediated cytoprotection. Transplantation 2002,74:315-9, incorporated herein in its entirety, with few modifications.

WT mice and Jα18^(−/−) mice, which lack type I NKT cells but have normallevels of type II NKT cells, (3-5 mice/group) or WT mice (2-3mice/group) treated 3 hours prior with 20 μg sulfatide/mouse wereanesthetized by intraperitoneal injection of 60 mg/kg sodiumpentobarbital. After midline laparotomy, an atraumatic clip was appliedto the hepatic triad (hepatic artery, portal vein, bile duct) of the 3cephalad liver lobes. The caudal lobes retained intact blood circulationto prevent intestinal venous congestion. The peritoneum was closed andmice were placed on a heating pad (˜37° C.). Ambient temperature rangedbetween 25-26° C. After 90 min of partial hepatic warm ischemia, theclip was removed, initiating reperfusion, and the abdominal wall wassutured. Mice were euthanized after 6 hours of reperfusion, and bloodand cephalad liver lobes were collected. Sham controls underwent thesame procedure but without vascular occlusion.

Cell preparation. Leukocytes were isolated from the cephalad liver lobesof the mice using mechanical crushing followed by Percoll gradientseparation and RBC lysis as described in Halder R C, Aguilera C, MaricicI, et al., Type II NKT cell-mediated anergy induction in type I NKTprevents inflammatory liver disease. J Clin Invest 2007, 117:2302-12.

Flow cytometry. Leukocytes were suspended in FACS buffer (PBS containing0.02% NaN₃ and 2% FCS), blocked (anti-mouse FcR-γ, BD Pharmingen, SanDiego, Calif.) and stained with loaded mCD1d-tetramer-PE or PE-, FTC-,or PE-Cy5-labeled anti-mouse antibodies (BD Pharmingen, San Diego,Calif. or eBioscience Inc., San Diego, Calif.) as indicated.Intracellular cytokine staining (ICCS) of liver mononuclear cells (MNCs)was carried out as described in Halder R C, Aguilera C, Maricic I, etal., Type II NKT cell-mediated anergy induction in type I NKT preventsinflammatory liver disease. J Clin Invest 2007, 117:2302-12. Analysiswas performed on a FACSCalibur instrument using CellQuest software(version 4.0.2, BD, Franklin Lakes, N.J.). Gr-1^(high) and Gr-1^(int)populations were gated. See FIG. 2 a.

Statistics. Data are expressed as mean t SEM for each group. P<0.005.Statistical differences between groups were evaluated by unpaired,one-tailed Student's t test using GraphPad Prism software (version 5.0a,GraphPad Software Inc., La Jolla, Calif.).

Results. The Gr-1^(int) subset of cells is comprised predominantly ofmonocytes and myeloid precursors. Following ischemia and reperfusioninjury (IRI), Gr-1^(int) cells were increased around 3.5-fold (p<0.005)compared to sham in the livers of WT mice, while no increase wasobserved following ischemia and reperfusion injury in Jα18^(−/−) mice.See FIG. 2 b , top panels. Therefore, the hepatic recruitment of myeloidcell subsets during ischemia and reperfusion injury is dependent on thepresence of type I NKT cells, which are lacking in Jα18^(−/−) mice.

When compared to untreated mice, the Gr-1^(int) cell subset was reducedin sulfatide-treated mice by ˜50% following IRI. This suggests that themyeloid cell recruitment activity of type I NKT cells was reducedfollowing sulfatide treatment.

The Gr-1^(high) cell subset, which mainly consists of granulocytes, didnot differ significantly between livers of sham controls and ischemiaand reperfusion injury-induced mice. FIG. 2 b , bottom panels.

Example 4

Sulfatide Administration Prior to Ischemia and Reperfusion InjuryInduction Significantly Inhibits IFN-γ Secretion by Type I NKT Cells.

One group of WT mice (n=3) was injected with sulfatide (20 μg/mousei.p.) 3 hrs prior to ischemia induction (Sulf. IRI), the other groups(IRI (n=3), sham (n=2)) were not pretreated. Hepatic ischemia andreperfusion injury (90 min of ischemia and 6 hrs of reperfusion) andsham surgery were performed as described in Example 3.

Cell preparation. Leukocytes were isolated from murine cephalad liverlobes, using mechanical crushing followed by Percoll gradient separationand RBC lysis as described in Halder R C, Aguilera C, Maricic 1, et al.,Type 11 NKT cell-mediated anergy induction in type I NKT preventsinflammatory liver disease. J Clin Invest 2007, 117:2302-12.

Flow cytometry. Leukocytes were suspended in FACS buffer (PBS containing0.02% NaN₃ and 2% FCS), blocked (anti-mouse FcR-γ, BD Pharmingen, SanDiego, Calif.) and stained with αGalCer loaded mCD1d-tetramer-PEanti-mouse antibodies (BD Pharmingen, San Diego, Calif. or eBioscienceInc., San Diego, Calif.). Analysis was performed on a FACSCaliburinstrument using CellQuest software (version 4.0.2, BD, Franklin Lakes,N.J.).

Statistics. Data are expressed as mean t SEM for each group. P<0.01.Statistical differences between groups were evaluated by unpaired,one-tailed Student's t test using GraphPad Prism software (version 5.0a,GraphPad Software Inc., La Jolla, Calif.).

Results. After ischemia and reperfusion injury induction, type I NKTcells show increased IFN-γ production compared to type I NKT cells fromsham surgery, while administration of sulfatide 3 hrs prior to ischemiaand reperfusion injury significantly reduced IFN-γ secretion by type INKT cells. See FIG. 2 c.

Example 5

Suppression of Type I NKT Cells Results in Reduced Hepatic NecrosisFollowing IRI

Purified bovine myelin-derived sulfatide (>90% pure), purchased fromMatreya Inc., Pleasant Gap, Pa., was dissolved in vehicle (0.5%polysorbate-20 (Tween-20) and 0.9% NaCl solution) and diluted in PBS.Groups of BU6 mice (WT sulfatide) and Jα18 mice (Jα18^(−/−) sulfatide)were treated with sulfatide (20 μg/mouse) intraperitoneally 3 to 48 hrsprior to ischemia induction or sham surgery. Hepatic ischemia andreperfusion injury and sham surgery were conducted as described inExample 3 on sulfatide treated and untreated WT and Jα18^(−/−) mice.Following 90 min of ischemia and 24 hrs of reperfusion, liver tissues(cephalad lobes) were fixed in 10% formalin, embedded in paraffin andsections were stained with hematotoxylin and eosin for histologicalanalysis (IDEXX Laboratories Inc., Westbrook, Me.).

WT mice pretreated with sulfatide (WT sulfatide) and Jα18^(−/−) micedeveloped only minimal or no hepatic necrosis after IRI, whereas largenecrotic areas were found in the cephalad liver lobes of the untreated(WT) mice. See FIG. 4 , top panels. Sham controls showed no necrosis.See FIG. 4 , bottom panels. The histological analysis indicated thatnecrosis following IRI is reduced in the livers of mice lacking type 1NKT cells (Jα18^(−/−) mice) and mice in which the activity of type 1 NKTcells is suppressed by sulfatide treatment.

Example 6

Induction of Alcoholic Liver Disease in WT and Type 1 NKT Cell-Depleted(Jα18−/−) Mice

Following acclimatization with nutritionally adequate Lieber-DeCarliliquid diet for a week, 6-8 week old male C57BL/6J mice (B6) and Jα18−/−mice, which lack type I NKT cells but not type 2 NKT cells, were givenfree access to a liquid diet containing 5% ethanol or an isocaloricdextrin maltose-containing diet (Bio-Serv, NJ). Precautions were takento prepare the diet fresh every day using autoclaved water in sterilizedfeeders, changing feed daily.

B6 and Jα18−/− mice receiving the ethanol-containing diet were separatedinto two groups, the chronic ethanol feeding group and the chronic plusbinge ethanol feeding group. Mice in the chronic ethanol feeding groupwere fed liquid diet containing 5% ethanol for up to 4-5 weeks. Mice inthe chronic plus binge ethanol feeding group were fed liquid dietcontaining 5% ethanol for 10 days followed by (on day 11) a single highdose of gavaged ethanol (5 g/kg body weight). Mice in the control groupwere gavaged with isocaloric dextrin maltose. Following gavage, micewere kept on a temperature controlled warm pad and monitored. Thoughinitially slow moving, most mice receiving the high dose of gavagedethanol regained normal behavior within hours. Mice were euthanized 8-9hr after gavage.

H&E staining was performed on Liver lobe tissue obtained fromethanol-fed male B6 and Jα18−/− mice following either 10 days or 4-5weeks of chronic ethanol feeding or chronic plus binge ethanol feeding.As shown in FIG. 9 a (top panels), histopathological analysis did notshow liver damage in either B6 or Jα18−/− mice subjected to chronicethanol feeding for 10 days. Further, no liver damage was observed byhistopathological analysis of the liver tissue of B6 and Jα18−/− micesubjected to chronic ethanol feeding for 4-5 weeks (data not shown).Histopathological analysis of liver tissue from B6 mice subjected tochronic plus binge ethanol feeding, however, showed significant damage.See FIG. 9 a , bottom left panel. While liver tissue from Jα18−/− micesubjected to chronic plus binge ethanol feeding showed relatively littledamage. See FIG. 9 a , bottom right panel.

Serum ALT levels were measured in control B6 and Jα18−/− mice andethanol fed B6 and Jα18−/− mice following either 10 days or 4-5 weekschronic ethanol feeding and chronic plus binge feeding. As shown in FIG.9 b , left panel, chronic ethanol did not lead to significant elevationof serum ALT levels. Serum ALT levels were significantly increased in B6mice subjected to chronic plus binge ethanol feeding, and were increasedto a lesser extent in Jα18−/− mice subjected to chronic plus bingeethanol feeding compared to control. See FIG. 9 b , right panel.

Maximum liver damage (compared to ethanol-free controls), as exemplifiedby histology or ALT/AST serum levels, was observed in B6 and Jα18−/−mice 6-8 hrs following high dose ethanol gavage. Between the groupsreceiving chronic plus binge ethanol feeding, Jα18−/− mice, which lacktype I NKT cells exhibited less liver damage.

Since chronic ethanol feeding for 10 days or for 4-5 weeks did not leadto any significant liver damage as exemplified by histopathologicalanalysis and no significant change in serum ALT/AST levels, this isreferred to pre or subclinical phase ALD. Clinical phase ALD was inducedfollowing a second step chronic-binge alcohol feeding.

Liver mononuclear cells (MNCs) were isolated from groups (4 in each) ofmale B6 and Jα18−/− mice following 5 weeks of feeding with either aliquid Lieber-Decarli diet containing 5% ethanol (chronic ethanolfeeding) or a control diet containing a similar number of calories. Theisolated liver MNCs were stained with various cell surface antibodiesand analyzed by flow cytometry. An accumulation of activated type I NKTcells and CD11b+Gr-1+ myeloid cells was observed in the livers of B6,but not Jα18−/−, mice in the preclinical ALD phase. See FIG. 5 .

Example 7

Prevention of Alcohol-Induced Liver Injury by Sulfatide or all-TransRetinoic Acid (ATRA)

Groups of 7-week-old B6 (WT) and Jα18−/− male mice were injected (i.p.)with: 20 micrograms/mouse sulfatide at days 1 and 10; 0.3milligrams/mouse ATRA at days 6-10, or vehicle/DMSO and were fed eithera liquid diet containing 5% ethanol for 10 days followed by (on day 11)a single high dose of gavaged ethanol (5 g/kg body weight) (chronic plusbinge ethanol feeding group) or an isocaloric dextrin maltose-containingdiet followed by an isocaloric gavage of with dextrin maltose (controlgroup). The mice were euthanized 6-8 hours following gavage and serumand liver tissue was harvested.

Serum ALT levels were determined for each group of mice. As shown inFIG. 6 , serum ALT levels were significantly increased in vehicle/DMSOinjected B6 mice subjected to chronic plus binge ethanol feeding (blackbar), while the serum ALT levels in chronic plus binge ethanol fed B6mice receiving either sulfatide (horizontally-striped bar) or ATRA(vertically-striped bar) injections were similar to those of control fedB6 mice (unshaded bar). Serum ALT levels were increased in vehicle/DMSOinjected Jα18−/− mice subjected to chronic plus binge ethanol feeding(black bar) compared to control fed Jα8−/− mice (unshaded bar), however,vehicle/DMSO injected Jα8−/− mice subjected to chronic plus bingeethanol feeding had reduced serum ALT levels compared to vehicle/DMSOinjected B6 mice subjected to chronic plus binge ethanol feeding. SeeFIG. 6 .

Histological examination for hepatic steatohepatitis (fatty liverdisease) was performed on liver tissue harvested from vehicle/DMSO,sulfatide or ATRA injected B6 mice following chronic plus binge ethanolfeeding. As shown in FIG. 7 , liver tissue from the vehicle/DMSOinjected B6 mice showed significant signs of damage following chronicplus binge ethanol feeding. Liver tissue obtained from sulfatide or ATRAinjected B6 mice, however, shows little sign of fatty liver disease.

Histological and serum liver enzyme analysis suggests thatadministration of either sulfatide or the retinoic acid receptor (RAR)agonist, ATRA, can protect against liver damage caused by excessivealcohol consumption.

Example 8

Inhibition of Type I NKT Cell Proliferation by ATRA

Type I NKT cells were isolated from the spleens of naïve B6 mice andcultured in vitro with [³H]-thymidine and an optimal concentration ofαGalCer alone, an optimal concentration of αGalCer plus 0.15 μg/ml, 1.2μg/ml, or 18.8 μg/ml ATRA, or ATRA alone. Proliferation of the type INKT cells was measured by [³H]-thymidine incorporation. As shown in FIG.8 a , cell proliferation was similar for type 1 NKT cells grown in 0.15μg/ml ATRA and αGalCer or αGalCer alone, while, by comparison,proliferation was reduced for type 1 NKT cells grown in 1.2 μg/ml or18.8 μg/ml ATRA and αGalCer. The greatest inhibition of αGalCer-inducedtype 1 NKT cell proliferation was observed for 18.8 μg/ml ATRA. No orminimal proliferation was observed for type 1 NKT cells grown in ATRAalone.

Example 9

Inhibition of Type I NKT Cell Activity by ATRA

The affect of ATRA on IL-2 cytokine secretion by a type I NKT cells inresponse to an in vitro challenge with αGalCer was tested byco-culturing NKT cells (Hy1.2) and irradiated antigen-presenting cells(APCs) in vitro with an optimal concentration of αGalCer in the presenceor absence of ATRA (0.15 μg/ml, 1.2 μg/ml, or 18.8 μg/ml) for a periodof 16 hours. Supernatants were collected and secreted IL-2 levels weremeasured using IL-2 sandwich ELISA.

IL-2 secretion levels were not significantly affected by 0.15 μg/mlATRA, however, significant reductions in IL-2 secretion was observed fortype 1 NKT cells cultured in the presence of 1.2 μg/ml or 18.8 μg/mlATRA. The greatest reduction in IL-2 secretion was achieved by 18.8μg/ml ATRA. See FIG. 8 b.

Functional changes in type I NKT cells following ATRA administrationwere further tested. In two independent experiments, groups of Black 6(WT) mice were injected intraperitoneally with 0.3 mg/animal (˜15 mg/Kgbody weight) ATRA or vehicle (DMSO) daily for a period of 5 days. Type INKT cells were purified and cultured in vitro with increasingconcentrations of αGalCer. Cell proliferation was measured andsupernatants were collected. The cytokine response of the isolated typeI NKT after in vitro challenge with αGalCer was determined by sandwichELISA for IFN-γ, IL-4, IL-6, IL-10, IL-12, and IL-13. The type I NKTcells isolated from ATRA injected mice did not proliferate in responseto αGalCer stimulation (data not shown). As shown in FIG. 8 c , type INKT cells isolated from vehicle (DMSO) injected mice secrete IFN-γ,IL-4, and IL-13 in response to αGalCer stimulation, but not IL-6, IL-10,or IL-12. No secretion of IFN-γ, IL-6, IL-10, IL-12, or IL-13 inresponse to αGalCer stimulation was detected from type I NKT cellsisolated from ATRA injected mice. Reduced levels of IL-4 secretion wereobserved from type I NKT cells isolated from ATRA injected mice inresponse to αGalCer stimulation. See FIG. 8 c.

Example 10

ATRA does not Inhibit Class II MHC-Restricted Conventional CD4 T Cells

The effect of ATRA on class II MHC-restricted conventional CD4 T Cellswas tested by isolating myelin basic protein MBPAc1-9-reactive CD4+ Tcells from naïve B10.PL VB8.2 TCR transgenic mice and culturing thecells in vitro with [³H]-thymidine and an optimal concentration ofMBPAc1-9 in the presence or absence of graded concentrations of ATRA(0.15, 1.2, 18.8 μg/ml). Proliferation of the MBPAc1-9-reactive CD4+ Tcells was measured by [³H]-thymidine incorporation. No inhibition ofMBPAc1-9-stimulated proliferation was observed in ATRA treated cultures.See FIG. 10 . This suggests that ATRA does not directly inhibit theactivity of MHC-restricted CD4+ T cells.

Example 11

ATRA Treatment in the Absence of Antigen-Presenting Cells Inhibits Type1 NKT Cell Function

The effect of ATRA treatment on type I NKT cells in the absence ofantigen-presenting cells was also measured. Type I NKT hybridoma cells(Hy1.2) were cultured in vitro either without ATRA or with 5 μg/ml, 10μg/ml, or 20 μg/ml ATRA for 24 hr. The cells were then washed threetimes and co-cultured with irradiated splenocytes (APCs) in the presenceof graded concentrations of the lipid antigen, αGalCer. Supernatantswere collected 16 hour later and IL-2 levels were measured using IL-2sandwich ELISA. As shown in FIG. 11 , treatment of type I NKT hybridomacells (Hy1.2) with ATRA inhibits IL-2 secretion. This suggests that ATRAtreatment in the absence of antigen-presenting cells inhibits theeffector function of type I NKT cells.

Example 12

Inhibition of Type I NKT Cell Activity by Subtype-Specific RAR Agonists

Retinoic acid receptors (RARs) comprise three major subtypes: RARα, RARβ, and RARγ. The RAR agonist ATRA is not selective for a specificsubtype. The contribution of RAR subtypes to inhibition of type I NKTcell activity was tested with subtype-selective RAR agonists as follows.Splenic cells were isolated from naïve B6 (WT) mice and cultured invitro in the presence of [³H]-thymidine, an optimal concentration ofαGalCer, and graded concentrations of the pan-RAR agonist ATRA, the RARαagonist AM580, the RAR β 2 agonist AC55649 or the RARγ agonist CD1530.Proliferation of the type I NKT cells in response to αGalCer stimulationwas measured by [³H]-thymidine incorporation.

Type I NKT cells cultured in the presence of ATRA, AM580, and CD1530exhibited similar levels of cell proliferation up to a concentration of10¹ μg/ml. See FIG. 12 . Cells cultured in the presence of 10¹ μg/mlATRA or CD1530 exhibited low or minimal proliferation, whileproliferation was maintained for cells cultured in 10¹ μg/ml AM580.Reduced cell proliferation compared to the other RAR agonists wasobserved for the RAR β 2 agonist, AC55649, at all concentrations tested,with the exception of 10¹ μg/ml, at which concentration low or minimalproliferation was observed for cells cultured in AC55649, ATRA orCD1530.

These results suggest that the RAR β 2 agonist, AC55649 is an effectiveinhibitor of type I NKT cell activity and that the retinoic acidreceptor-82 (RAR-β 2) signaling pathway is involved in the inhibition oftype I NKT cells.

Example 13

Effect of PPAR-γ Pathway Modulation on Type I NKT Cells

Peroxisome proliferator-activated receptors (PPARs) are ligand-inducibletranscription factors that belong to the nuclear hormone receptorsuperfamily, which also includes the receptors for thyroid hormone,retinoids, steroid hormones and vitamin D. PPARs regulate geneexpression by binding with Retinoid X Receptor (RXR) as a heterodimericpartner to specific Peroxisome Proliferator Response Elements (PPREs) inthe DNA. PPARs have been implicated as playing a role in lipid andenergy metabolism, inflammation, embryo implantation, diabetes andcancer.

The role of the PPAR-γ pathway in the inhibition of type I NKT cells wastested by culturing freshly isolated splenic type I NKT cells with[³H]-thymidine and an optimal concentration of αGalCer in the presenceor absence of graded concentrations of the PPAR-γ agonist rosiglitazoneor the PPAR-γ antagonist GW9662. Proliferation of the splenic type I NKTcells in response to αGalCer stimulation was quantified by[³H]-thymidine incorporation. Similar levels of proliferation wasobserved for cells cultured in either the PPAR-γ agonist, rosiglitazone,or the PPAR-γ antagonist. GW9662, at all concentrations. See FIG. 13 .These results suggest that specific inhibition or activation of thePPAR-γ pathway does not directly inhibit type I NKT cell proliferation.

Example 14

Examination of the Effect of Retinol Analogs on Type I NKT Cell Activity

Splenic cells were isolated from naïve B6 mice and cultured in vitrowith [³H]-thymidine and an optimal concentration of αGalCer in thepresence or absence of graded concentrations of ATRA, Retinol, 9-cis-RAor 13-cis-RA. Proliferation of the splenic type I NKT cells in responseto αGalCer stimulation was quantified by [³H]-thymidine incorporation.As shown in FIG. 14 , no or minimal proliferation was observed for cellscultured in 10¹ μg/ml ATRA, while the same level of inhibition of type INKT cell proliferation was only observed at concentrations of Retinol,9-cis-RA or 13-cis-RA which exceeded 10¹ μg/ml.

Example 15

Examination of the Effect of RAR Agonists on Type I NKT Cell Activity

In vitro proliferation and cytokine release assays were performed onfreshly isolated splenocytes and type I NKT cell hybridomas (1.2 Hyb).The cells were cultured in the presence of [3H]-thymidine and an optimalconcentration of αGalCer (10 ng/ml) with titrating concentrations ofATRA, Tretinoin, the RARα agonist AM580, the RAR β agonist AC55649 orthe RARγ agonist CD1530. Proliferation of the type I NKT cells inresponse to αGalCer stimulation was quantified by [³H]-thymidineincorporation. IL-2 levels were measured using IL-2 sandwich ELISA.Optimal concentrations of the RAR agonists was determined. A comparisonof the effect of the RAR agonists at their optimum concentrations isshown in FIG. 15 . The lowest levels of proliferation were observed forcells cultured in the presence of ATRA, Tretinoin, the RARγ agonistCD1530. Cells cultured in the presence of the RARγ agonist CD1530 showedthe lowest levels of IL-2 secretion. These results indicate that theRARγ agonist CD1530 is an effective inhibitor of type I NKT cellactivity.

Example 16

Comparison of Tazarotene and ATRA Inhibition of Type I NKT Cells

The effect of the selective RARγ agonist, Tazarotene, on type I NKT cellactivity was examined by in vitro proliferation and cytokine releaseassays.

In vitro proliferation and cytokine release assays were performed onfreshly isolated splenocytes and type I NKT cell hybridomas (1.2 Hyb).The cells were cultured in the presence of [³H]-thymidine and an optimalconcentration of αGalCer (10 ng/ml) with increasing concentrations ofATRA or Tazarotene. Proliferation of the type I NKT cells in response toαGalCer stimulation was quantified by [H]-thymidine incorporation. IL-2levels were measured using IL-2 sandwich ELISA. A comparison of type INKT cell inhibition by ATRA and Tazarotene is shown in FIG. 16 . Whileby RAR agonists inhibit type I NKT cell proliferation and IL-2secretion, Tazarotene achieves its inhibitory effects at lowerconcentrations. See FIG. 16 .

Example 17

Inhibition of Type I NKT Cells Following In Vivo Administration ofTazarotene and ATRA

Functional changes in type I NKT cells following in vivo administrationof Tazarotene or ATRA was tested. Groups of mice were injectedintraperitoneally with 300 g ATRA (15 mg/kg), 300 μg Tazarotene (15mg/kg) or vehicle (DMSO) daily for a period of 5 days. Splenic Type INKT cells were purified and cultured in vitro with increasingconcentrations of αGalCer. Cell proliferation was measured byquantification of [³H]-thymidine incorporation and the results are shownat FIG. 17 . A comparison of the proliferative response of cellsisolated from ATRA injected, Tazarotene injected, or vehicle (DMSO)injected mice to αGalCer stimulation, indicates that in vivoadministration of either ATRA or Tazarotene inhibits type I NKT cellactivity. The lowest levels of proliferation, however, were observed fortype I NKT cells isolated from Tazarotene injected mice. See FIG. 17 .

Mononuclear cells were isolated from livers of mice injected with eitherATRA, Tazarotene or vehicle (DMSO) and stained withαGalCer/CD1d-tetramer and a pan anti-TCRβ antibodies. Flowcytometry wasperformed and the population of αGalCer/CD1d-tetramer/TCRβ expressingcells (type I NKT cells) was quantified as shown in FIG. 18 . Nosignificant difference in the numbers of type I NKT cells in the liversof control vs. ATRA or Tazarotene administered animals was observed. SeeFIG. 18 . These results indicate that ATRA or Tazarotene administrationdoes not deplete liver type I NKT cells.

Prophetic Example 18

Prevention of ALD by RAR Agonist Administration

The effectiveness of RAR Agonists in the prevention or mitigation ofalcoholic liver disease is tested by injecting groups of mice with aneffective amount of: ATRA, Tazarotene, Tretinoin, the RARα agonistAM580, the RAR β agonist AC55649, the RAR agonist CD1530, orvehicle/DMSO daily for a period of 5 days during which time the mice areeither a liquid diet containing 5% ethanol for 10 days followed by (onday 11) a single high dose of gavaged ethanol (5 g/kg body weight)(chronic plus binge ethanol feeding group) or an isocaloric dextrinmaltose-containing diet followed by an isocaloric gavage of with dextrinmaltose (control group). The mice are euthanized 6-8 hours followinggavage and serum and liver tissue is harvested.

Serum ALT levels are determined for each group of mice. Serum ALT levelsare expected to be significantly increased in vehicle/DMSO injected micesubjected to chronic plus binge ethanol feeding compared to control fedmice. Chronic plus binge ethanol fed Mice receiving injections of ATRA,Tazarotene, Tretinoin, AM580, AC55649, CD1530 are expected to havereduced serum ALT levels compared to vehicle/DMSO injected micesubjected to chronic plus binge ethanol feeding. The serum ALT levels ofATRA Tazarotene, or Tretinoin injected mice subjected to chronic plusbinge ethanol feeding are expected to be similar to those observed incontrol fed mice. Histological examination for hepatic steatohepatitis(fatty liver disease) is expected to show significant damage of theliver tissue harvested from vehicle/DMSO injected mice following chronicplus binge ethanol feeding, while reduced or no damage is expected forliver tissue harvested from ATRA, Tazarotene, Tretinoin, AM580, AC55649,or CD1530 injected mice following chronic plus binge ethanol feeding.

Prophetic Example 19

Adoptive transfer experiments are used with αGalCer/CDd-tetramer-purified type I NKT cells from ATRA-treated orTazarotene-treated BU6 mice to examine whether a graded number of thesecells when transferred into nave BU6 recipients can inhibit liver injuryfollowing chronic plus binge ethanol feeding.

Purified (sorted) sulfatide-CD1d-tetramer+ T cells are tested todetermine if they can prevent ALD upon adoptive transfer into CD1d+/+and CD1d−/− mice. Cytokine knockout mice are used as donors ofsulfatide-reactive T cells to directly determine the role of IFN-γ, IL-4and IL-10 secretion by type II NK T cells in the regulation of ALD.Sulfatide-reactive T cells are isolated using tetramers and gradednumbers (0.5-1 million) are adoptively transferred into CD1d+/+ andCD1d−/− C57BL/6 recipients. Around 1.5 million sulfatide-CD1d-tetramer+cells are isolated from 18 naïve C57BL/6 mice. One day later recipientsare exposed to chronic plus binge ethanol feeding. For analyzing theroles of cytokine secretion by type II NKT cells, initially the IFN-γ,IL-4−/− or IL-10−/− are used on the BU6 background (Jackson Lab). Groupsof BU6 wild type, or knockout mice are injected with sulfatide (20μg/animal) and then ALD induced. In the absence of these type 2cytokines, sulfatide administration is expected to prevent ALD. Adoptivecell-transfer experiments are further performed withsulfatide-CD1d-tetramer+ cells from specific cytokine knockout mice,where graded numbers (0.5-1 million) are transferred into CD1d−/−C57BL/6 mice. One day later recipient mice are kept on ethanol feeding.In parallel, sulfatide-CD1d-tetramer+ T cells are transferred fromwild-type mice into CD1d+/+ BU6 mice as a positive control. Theseexperiments are expected to confirm directly the role of these cytokinessecreted by sulfatide-reactive T cells in the control of ALD.

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the present embodiments. Theforegoing description details certain preferred embodiments anddescribes the best mode contemplated by the inventors. It will beappreciated, however, that no matter how detailed the foregoing mayappear in text, the present embodiments may be practiced in many waysand should be construed in accordance with the appended claims and anyequivalents thereof.

What is claimed is:
 1. A method of inhibiting pro-inflammatory type INKT cells in a subject, comprising administering an RARγ-selectiveagonist, wherein the administered RARγ-selective agonist inhibitsactivation of pro-inflammatory type I NKT cells in the subject.
 2. Themethod of claim 1, wherein the RARγ-selective agonist is ATRA, retinol,9-cis-RA, 13-cis-RA, tretinoin, AM580, AC55649, CD1530, or Tazarotene.3. The method of claim 2, wherein the RARγ-selective agonist isTazarotene.
 4. The method of claim 1, wherein the amount of theRARγ-selective agonist is about 1 mg/day to about 10 mg/day.
 5. A methodof treating an inflammatory condition of fibrosis in a subject in needthereof, the method comprising administering an amount of RARγ-selectiveagonist sufficient to inhibit activation of pro-inflammatory type I NKTcells to the subject, wherein the fibrosis is associated with anincrease in pro-inflammatory type I NKT cells and the administeredRARγ-selective agonist inhibits activation of pro-inflammatory type INKT cells in the subject, thereby treating the inflammatory condition offibrosis.
 6. The method of claim 5, wherein the administering is by oraldelivery.
 7. The method of claim 5, wherein the administering is bypulmonary delivery.
 8. The method of claim 5, wherein the RARγ-selectiveagonist is ATRA, retinol, 9-cis-RA, 13-cis-RA, tretinoin, AM580,AC55649, CD1530, or Tazarotene.
 9. The method of claim 8, wherein theRARγ-selective agonist is Tazarotene.
 10. The method of claim 5, whereinthe amount of the RARγ-selective agonist is about 1 mg/day to about 10mg/day.